Target RNAs Strike Back on MicroRNAs

Abstract

MicroRNAs are extensively studied regulatory non-coding small RNAs that silence animal genes throughout most biological processes, typically doing so by binding to partially complementary sequences within target RNAs. A plethora of studies has described detailed mechanisms for microRNA biogenesis and function, as well as their temporal and spatial regulation during development. By inducing translational repression and/or degradation of their target RNAs, microRNAs can contribute to achieve highly specific cell- or tissue-specific gene expression, while their aberrant expression can lead to disease. Yet an unresolved aspect of microRNA biology is how such small RNA molecules are themselves cleared from the cell, especially under circumstances where fast microRNA turnover or specific degradation of individual microRNAs is required. In recent years, it was unexpectedly found that binding of specific target RNAs to microRNAs with extensive complementarity can reverse the outcome, triggering degradation of the bound microRNAs. This emerging pathway, named TDMD for Target RNA-Directed MicroRNA Degradation, leads to microRNA 3'-end tailing by the addition of A/U non-templated nucleotides, trimming or shortening from the 3' end, and highly specific microRNA loss, providing a new layer of microRNA regulation. Originally described in flies and known to be triggered by viral RNAs, novel endogenous instances of TDMD have been uncovered and are now starting to be understood. Here, we review our current knowledge of this pathway and its potential role in the control and diversification of microRNA expression patterns.

Keywords: Argonaute; TDMD; degradation; exoribonuclease; microRNA; tailing and trimming; terminal nucleotidyl transferase; uridylation.

FIGURE 1

Target binding architectures define the outcomes of miRNA binding. (A) Properties of TDMD-inducing target RNAs bound to their cognate miRNAs and concomitant miRNA tailing and trimming species accumulating during TDMD are schematically represented. Substoichiometric target RNA concentrations may suffice to trigger TDMD, allowing miRNAs to induce only limited or no target degradation. (B) Properties of canonical miRNA targets typically leading to target RNA silencing upon miRNA binding. Additional architectures such as ‘centered’ or ‘seedless’ interactions have been omitted, though the latter has been linked to TDMD (see text and Figure 2B).

FIGURE 2

Architecture of TDMD-inducing miRNA binding sites described in the literature. (A) Seed containing miRNA binding sites within artificially designed or naturally occurring target RNAs—either of viral or cellular origin. (B) Seedless miRNA binding site present in an artificial target RNA shown to elicit TDMD in vitro (Park et al., 2017). Dashes represent Watson–Crick base pairs and dots represent Wobble base pairs.

FIGURE 3

Mechanism of TDMD. (A, Left) Proposed models for TDMD. In tailing-dependent miRNA degradation, highly complementary target RNAs would either expose the miRNA 3′ ends to TNTases by detaching them from AGO’s 3′-end binding pocket in the PAZ domain (see panel B), favor the kinetics of TNTases on the 3′ ends of the bound miRNAs, or both. The tailed species would subsequently serve as preferred substrates for 3′-to-5′ exoribonucleases. Alternatively, tailing might promote miRNA unloading from AGO for subsequent degradation of free miRNAs in the cytoplasm. In tailing-independent miRNA degradation, highly complementary target RNA would promote unloading of miRNAs from AGO, leading to subsequent degradation of free miRNAs in the cytoplasm. Increased tailing could be a parallel process, rather than a cause of degradation, induced by highly complementary target RNAs binding to miRNAs. Tailing could in turn have either stabilizing effects or an influence on the activity of miRNAs. (Right) In plants, 2′-O-methylation at the 3′ end of miRNAs prevents 3′ tailing and stabilizes the modified miRNAs (see main text). (B) Structural rearrangements induced on AGO by binding of target RNAs with different architectures. (Top left) Binary complex of a miRNA loaded on AGO; the miRNA 5′ end is bound to the MID domain while the 3′ end is bound to the PAZ domain and therefore protected from terminal modifications. (Top right) Ternary complex comprising an AGO-loaded siRNA (or miRNA) bound to a perfectly complementary target RNA; the extensive 3′ pairing releases the 3′ end of the siRNA and induces a conformational change that switches the complex from an inactive to an active slicing conformation, cleaving the target at the catalytic site (solid arrowhead). The rapid release of the cleaved target would preclude tailing of the AGO-bound miRNA, which would return to its protected conformation. (Bottom left) Ternary complex comprising an AGO-loaded miRNA bound to a canonical seed-matched target RNA. Due to lack of pairing to the 3′ end of the miRNA, the 3′ end would remain bound and protected within the AGO PAZ domain. (Bottom right) Hypothetical structure of a ternary complex comprising an AGO-loaded miRNA bound to a TDMD-competent target RNA; the extensive 3′ pairing might expose the 3′ end in a conformation that would render it susceptible to attack by modifying enzymes (e.g., TNTases).

FIGURE 4

Functional implications of TDMD. TDMD might control and diversify miRNA expression patterns by means of: (A) allowing expression of specific miRNA family members from single gene clusters, e.g., in a tissue- or developmental time-specific manner; (B) promoting the recycling of Argonaute (Ago) proteins and thus the loading of newly synthesized miRNAs; or (C) Right panel: avoiding miRNA silencing in cis, e.g., under circumstances where TDMD-competent target RNAs might be too scarce to globally affect miRNA abundance in trans, but effective enough to trigger TDMD in cis and becoming immune to miRNA silencing (in contrast to the left panel where an abundant target RNA would additionally deplete a specific miRNA in trans globally in the cell, as in A). Shine emphasis is shown on “TDMD-primed” Ago-miRNA-target complexes.